As part of a continuing study of the neurophysin-peptide hormone system, the tripeptide analogue of vasopressin, L-methionyl-L-tyrosyl-L-phenylalaninamide, has been modified for use in photoaffinity labeling studies of the hormone binding site on neurophysin. The photo-sensitive peptide, L-methionyl-L-tyrosyl-p-azido-L-phenylalaninamide, was found by equilibrium dialysis in the absence of light to be reversibly bound to bovine neurophysin II. The dissociation constant, stoichiometry, and pH-dependence indicate that the interaction is similar to the interaction of hormone with neurophysin. The affinity reagent also competitively inhibits binding of L-methionyl-L-tryosyl-L-phenylalaninamide which has been shown to competitively inhibit binding of hormone by neurophysin. Exposing the noncovalent reagent-neurophysin complex to ultraviolet radiation leads to formation of a covalent complex in moderate yields. Non-neurophysin proteins are covalently labeled to only a few percent of the neurophysins. The covalent complex has been derivitized and fragmented and will be subjected to peptide mapping and sequencing in an attempt to determine which residues of the protein are found near or in the binding site. Covalent labeling of all species of neurophysin studied to date proceeds to a similar extent.